Mass Spectrometry-Based Redox and Protein Profiling of Failing Human Hearts

Abstract

Oxidative stress contributes to detrimental functional decline of the myocardium, leading to the impairment of the antioxidative defense, dysregulation of redox signaling, and protein damage. In order to precisely dissect the changes of the myocardial redox state correlated with oxidative stress and heart failure, we subjected left-ventricular tissue specimens collected from control or failing human hearts to comprehensive mass spectrometry-based redox and quantitative proteomics, as well as glutathione status analyses. As a result, we report that failing hearts have lower glutathione to glutathione disulfide ratios and increased oxidation of a number of different proteins, including constituents of the contractile machinery as well as glycolytic enzymes. Furthermore, quantitative proteomics of failing hearts revealed a higher abundance of proteins responsible for extracellular matrix remodeling and reduced abundance of several ion transporters, corroborating contractile impairment. Similar effects were recapitulated by an in vitro cell culture model under a controlled oxygen atmosphere. Together, this study provides to our knowledge the most comprehensive report integrating analyses of protein abundance and global and peptide-level redox state in end-stage failing human hearts as well as oxygen-dependent redox and global proteome profiles of cultured human cardiomyocytes.

Keywords: cardiomyopathy; failing hearts; oxidative stress; redox proteomics.

Figure 1

Failing hearts display a significantly reduced GSH/GSSG ratio (left). Absolute GSH levels (middle) in failing hearts are slightly reduced (not significant, p = 0.313), while GSSG content (right) is slightly increased in failing (not significant, p = 0.194). Values represent mean values and S.E.M. n (control) = 10, n (Failing hearts) = 15. ** Student t-test p-value < 0.01; ns—not significant.

Figure 2

Heatmap of significantly more oxidized-cysteine residues in failing hearts. Significantly more oxidized cysteine-containing peptides p-value < 0.05, fold-change (Cys_red/Cys_ox ratio of control versus failing heart >1.35) in failing hearts are labeled with gene names of their corresponding proteins and the cysteine position in the amino acid sequence. Colors of the map illustrate a degree of oxidation: the red color indicates lower Cys_red/Cys_ox ratio, depicting higher degree of oxidation. On the contrary, the blue color represents higher Cys_red/Cys_ox ratio and therefore indicates lower oxidation state of the cysteine.

Figure 3

Gene-ontology-enrichment analysis of biological processes (GOBP) of corresponding proteins from significantly more oxidized cysteine-containing peptides in failing hearts as the input, revealing higher oxidation of proteins of contractile machinery and glucose metabolism. ATP—adenosine triphosphate, NAD(H)—nicotinamide adenine dinucleotide (reduced).

Figure 4

Proteomic analysis revealed altered protein expression patterns in failing hearts. (A): Volcano plot of proteins identified in failing and control hearts. Significant hits are labeled with their corresponding gene names and depicted in black. Proteins significantly more expressed in failing hearts are shown on the right while the proteins more abundant in the controls are located on the left side of the plot. (B): String protein-interaction analysis of proteins significantly more expressed in failing hearts results in two differential protein clusters (false discovery rate (FDR)-corrected p-value < 0.05, fold change (failing/control hearts) >1.5). (C): Bar plot displaying fold changes of significantly more expressed proteins from the lower String protein cluster (values normalized on the mean of the control group). (D): Bar plot representing fold changes of significantly up-regulated proteins from the upper String protein cluster, namely involved in extracellular matrix remodeling, wound healing, and fibrosis (values normalized on the mean of the control group). PEDF—pigment epithelium-derived factor (gene name: SERPINF1), BP—binding protein.

Figure 5

AC16 cells cultured under higher oxygen concentration (higher oxidative stress) display some similar protein signatures as failing human hearts. (A). Volcano plot of differentially expressed proteins between normoxic (N) and physioxic conditions (harvested under physioxia, PP); (B). Volcano plot of differentially expressed proteins between cells cultured in physioxia but harvested in normoxia (PN) and cells cultured and harvested under physioxia (PP). (C). Gene-ontology enrichment of biological process (GOBP) with significantly more abundant proteins in normoxia (N) compared to physioxia (PP) (proteins used for input must have passed the significance threshold of false discovery rate (FDR)-corrected p-value < 0.05 and have a fold change (compared to physioxia) >1.3). N = 3 per condition, FDR.

Figure 6

Proteins with a significantly lower expression in failing hearts compared to nonfailing control samples. Bar plot representing fold change in the expression of significantly less expressed proteins in failing hearts (Figure 4A, left side of the volcano plot) which include several metabolic enzymes as well as proteins implicated in protein translation and ion transport (FDR-corrected p-value < 0.05, fold change (failing/control hearts) <0.7).